The long term goal of this research project is to elucidate the roles of Ku protein in the response of cells to stress, specifically to heat shock and to ionizing radiation During the next funding period, there are three aims. Specific Aim I focuses on the molecular mechanism(s) by which Ku suppresses heat-induction of hsp70. We will test a hypothesis, inferred from our preliminary studies, that Ku70 overexpression leads to hypermethylation of CpGs within the hsp70 promoter, and that this hypermethylation in turn suppresses thermal induction of hsp70. First, bisulfite genomic sequencing and genomic footprinting of the hsp70 promoter will be carried out to determine how the CpG methylation and protein-binding patterns change in response to Ku70 and/or Ku80 overexpression. Second, the effect of DNA demethylation agent 5-AzaC on the heat-induction of hsp70 mRNA and protein, and on the CpG methylation and protein-binding patterns of hsp70 promoter in control and Ku70-overexpressing cells will be determined, both before and after heat shock. These experiments aim to establish a firm link between methylation of specific CpGs of the hsp70 promoter by Ku-overexpression and the suppression of hsp70 thermal induction. Third, electrophoretic mobility shift assays and transient transfection experiments, using oligomeric and reporter-linked DNAs (covering the cis-elements within the hsp70 promoter) with specific 5-methylcytosine substitutions, will also be performed to correlate CpG methylation, protein-binding, and hsp70 promoter transactivation. In Specific Aim II, to study and dissect the roles of various domains of Ku70 and Ku80 in cellular response to heat shock and to ionizing radiation, we have generated rodent cell lines stably and constitutively overexpresssing intact or various domains of Ku70 and Ku80. In addition, we will take advantage of the cell lines lacking Ku70, Ku80, or both Ku70 and Ku80, which we have recently established, to express intact Ku or various Ku fragments, either individually or in combinations. Using these cell lines, we will determine which domains of Ku70 and Ku80 are involved in modulating cellular responses to heat shock and ionizing radiation. In Specific Aim III, using recombinant adeno- and adeno-associated viral vectors, that express Ku antisense RNA or dominant negative Ku gene fragments, we will evaluate whether down-regulation of Ku level and/or activity can enhance the radiosensitivity of tumor cells in vitro and in vivo. The information gained will serve as a guide in the design of new strategies for sensitizing tumor cells to ionizing radiation.